Human-human hybridom for neoplasms CLNH5 and CLNH11 specific antibody compositions

ABSTRACT

CLNH5 and CLNH11 specific hybridomas, human monoclonal antibodies and their uses are provided. The antibodies distinguish a human neoplastic cell from a normal cell of the same tissue type. The monoclonal antibodies find use in therapy and diagnosis, both in vitro and in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.08/163,377, filed Dec. 7, 1993, now abandoned which is a divisional ofU.S. application Ser. No. 07/113,212, filed Oct. 23, 1987, now U.S. Pat.No. 5,286,647, which is a continuation-in-part of 06/573,974, filed Feb.21, 1984, now abandoned, which is a U.S. National Phase ofPCT/US83/00781, filed on May 20, 1983, which claims priority to JapaneseApplication No. JP84843-3 and is a continuation-in-part of U.S.application Ser. No. 06/465,081, filed Feb. 9, 1983, which issued asU.S. Pat. No. 4,618,577.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The mammalian immune system has a matchless ability to produce moleculeswith specificity and avidity for a particular spatial and polarstructure, as may be found with sequences of amino acids and sugars. Fora long period of time, one was dependent upon producing antibodiesemploying the immune system in vivo. The resulting polycolonalantibodies demonstrated high specificity for a specific antigen, butcould not discriminate between various sites on the antigen and,furthermore, were a mixture of antibodies of varying specificity andavidity. Thus, one observed the averaging over the entire compositionand not the properties of a specific antibody.

With the seminal discovery by Milstein and Kohler, one can now producehomogeneous compositions of antibodies by fusing a B-lymphocyte with amyeloma cell to produce a cell referred to as a hybridoma. For the mostpart, the use of this technology has been limited to mouse cells, wherestable myeloma lines have served as fusion partners to provide stablehybridomas which can be produced with high efficiency and are capable ofbeing maintained as productive entities over long periods of time.Higher organisms, particularly humans, have proven to be much moreintractable in developing fusion partners and hybridomas. However, in1980, the first human fusion partner was reported by Drs. Olsson andKaplan and since that time, an additional few human fusion partners havebeen reported. Nevertheless, the preparation of hybridomas byhuman-human crosses has remained difficult due to problems of efficiencyin fusion, culturing the cells, and maintaining their productivecapabilities. However, because of the many advantages of having humanhybridomas which produce antibodies allogenic to a human host,particularly for in vivo applications, human hybridomas remain of greatinterest. In other instances, even with the difficulties encounteredwith human-human crosses, the human hybridoma may be preferable to aheterogenic cross, where the resulting hybridoma may lose the geneticinformation for the monoclonal antibodies (MoAbs) after a number ofpassages.

One of the areas of interest for the use of monoclonal antibodies is indiagnosing and treating cancer. Monoclonal antibodies for these purposesdesirably are specific for a particular type of cancer or subset ofcancers, rather than being specific for a particular host cancer cell.It is therefore desirable to develop monoclonal antibodies which can beused in the diagnosis and treatment of human cancers.

2. Relevant Literature

Nowinski et al., Science (1980) 210:537-539 describe human monoclonalantibodies against Forssman antigen. Croce et al., Nature (1980)288:488-489 describe human hybridomas secreting antibodies to measlesvirus. Olsson and Kaplan, PNAS USA (1980) 77:5429-5431 describehuman-human hybridomas producing monoclonal antibodies of predefinedantigenic specificity as well as the fusion partner employed forproduction of the antibodies. See also copending application Ser. No.247,652, filed Mar. 26, 1981.

Schlom, PNAS USA (1980) 77:6841-6845 describes monoclonal antibodies forbreast cancer and Sikora, Brit. J. of Cancer (1981) 43:696 describesseparating in situ lymphocytes from a cancer providing antibodiesspecific for the cancer. In the Proceedings of the 15th LeukocyteCulture Conference, Parker and O'Brien, eds., Wiley Interscience, N.Y.,Dec. 5-10, 1982, the subject hybridoma is described. This abstract isincorporated herein by reference.

SUMMARY OF THE INVENTION

Lymphocytes derived from a neoplastic human host are immortalized byfusion with human fusion partners to provide human×human hybridomassecreting monoclonal antibodies (MoAbs) specific for a cell surfaceantigen of a neoplastic cell. Particularly, monoclonal antibodiesspecific for antigens of solid tumor cells such as cervical cancer cellswhich are not found on normal cells of the same tissue type are providedfor use in diagnostics and therapy.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Human monoclonal antibodies (MoAbs) specific for antigens found on thesurface of neoplastic cells from solid tumors are obtained fromhuman×human fusions employing B lymphocytes, e.g., from lymph nodesdraining a solid tumor. Particularly, lymph nodes are selected whichappear to be active based on necrosis of tumor cells in the vicinity ofthe lymph node in an immunocompetent host.

The draining lymph node(s) may be isolated in conjunction with a varietyof human tissue, e.g., cervix, mammary, colon, lungs, prostate, skin,etc.

The fusion partner may be any convenient immortalized human B-cell whichdoes not secrete immunoglobulins or individual chains or fragmentsthereof, which can be selected against, as with HAT medium, anddesirably which has a high fusion efficiency. Illustrative fusionpartners are UC729-6, J-4 (SKO-007), and GM1500 6TG-A12.

The fusions may be performed as described in the literature employingPEG1500 as fusogen, plating the cells in HAT medium in a plurality ofwells and then screening supernatants in the viable cell-containingwells for antibodies of interest. Wells positive for reactivity are thencloned by limiting dilution and expanded.

The hybridomas and monoclonal antibodies of this invention can find usein a variety of ways, particularly as sources of genetic material, asreagents, and as precursors to products which find use as reagents.

The subject hybridomas may be used as a source of genetic material. Forexample, the subject hybridomas may be fused with other fusion partnersto provide novel hybridomas having the same secretory capabilities asthe hybridomas providing the genetic material to provide antibodieshaving the same specificity. Such fusions may result in the productionof antibodies having different heavy chains so as to provide the otherclasses or subclasses of antibodies, e.g., G, A, or M.

The monoclonal antibodies can be used in a variety of ways, both in vivoand in vitro diagnosis, as well as in therapy. For many applications,the antibodies will be labeled with a compound which imparts a desiredproperty to the antibodies, such as providing a detectable signal,providing cytotoxicity, providing for localized electromagneticradiation, or the like. Labels may include radionuclides, enzymes,fluorescers, toxins or the cytotoxic fragment of toxins, particles,metals, metalloids, etc. The antibodies may be incorporated in liposomemembranes or modified with lipids, so as to be incorporated in suchmembranes. The antibodies by themselves or labeled, may be used in invitro diagnosis for measuring the presence of antigens associated with aneoplasm such as cervical cancer, for in vivo diagnosis for introductioninto a host, e.g., intravenously in a physiologically acceptablecarrier, e.g., PBS, or may be introduced for therapeutic purposes in thesame manner.

The antibodies by themselves or labeled, may also be used for treating aneoplasm in human host such as cervical carcinoma, prostate tumor, coloncarcinoma, lung cancer, breast cancer or melanoma. The antibodies ofthis invention are easily soluble in physiological saline, and thereforecan be injected intravenously or intramuscularly as a saline solution ora drip. Furthermore, the antibodies of the invention can be used in theform of an ointment or suppository.

The amount of antibody employed will vary depending upon the particularapplication. Introduction of antibodies for diagnostic and therapeuticpurposes has been extensively described in literature.

The entire antibody need not be used, for many applications only afragment having intact variable regions will suffice. For example, Fabfragments, F(ab')₂ fragments, or Fv fragments may suffice. Additionally,chimeric antibodies having the variable regions of the instantantibodies can be produced.

Exemplary of human-human hybridomas utilizing lymphocytes from draininglymph nodes are the novel hybridomas CLNH5 and CLNH11, hybridomasobtained from CLNH5 and 11, antibodies derived from such hybridomas,derivatives of such antibodies and the use of the antibodies and theirderivatives for diagnosis and therapy. CLNH5 and 11 are obtained byfusion between the fusion partner UC729-6 with lymphocytes from lymphnode cells of a patient having cervical cancer. Hybridoma CLNH11 wasdeposited with the American Type Culture Collection on Jun. 13, 1983,with Accession number HB 8307. UC729-6 is on deposit at the A.T.C.C.with Accession No. CRL 8061. UC729-6 was deposited for patent purposesin conjunction with the filing of application Ser. No. 247,652.

The lymphocytes employed for that fusion were from a draining lymph nodefrom a cervical carcinoma and peripheral blood lymphocytes from apatient having cervical carcinoma. The fusion was performed by combiningthe patient's lymphocytes from the lymph node with the fusion partnerUC729-6 at a ratio of about 2:1 in a solution of about 35% polyethyleneglycol in HEPES buffered RPMI 1640. The mixture of cells was thensuspended in appropriate selective medium, particularly HAT mediumcontaining about 10% fetal bovine serum, placed in wells at about 10⁵cells per well and a sufficient time permitted for the cells to grow.The selective medium was replaced from time to time.

Wells from the above fusion provided clones specifically reactive withcell surface antigens of the cervical cancer cells of the host patientwhich were designated CLNH5 and 11. These wells provided human IgM andIgG monoclonal antibodies, respectively, which react with antigen foundon a variety of cervical carcinomas and other tumor cell lines, e.g.,small cell carcinoma of the lung, but not with normal tissues and normalcell lines, which were tested.

Additional fusions of lymphocytes from a lymph node draining a solidtumor were performed using lymph nodes from a patient with vulvarcarcinoma and from a patient with a Wilm's tumor. In each fusion, anumber of hybridomas secreting antibodies which reacted with cellsurface antigens found on carcinoma cells and either not detectable onnormal cells or found on normal cells in significantly reduced numberswere produced. Exemplary hybridomas are described in detail in theexperimental section.

The fusion frequency for human lymphocytes from lymph nodes is higherthan the fusion frequency using peripheral blood lymphocytes (1.9 incomparison to 0.51 hybrid+wells/10⁶ lymphycytes mean fusion rate; SeeGlassy et al., p. 211-225 in Human Hybridomas and Monoclonal Antibodies,Plenum Publishing Corp. 1985, Engleman et al., eds.). In addition to theenhanced fusion frequency, it has been found that hybridomas can beselected from fusions of lymphocytes from draining lymph nodes whichsecrete antibodies that bind to antigens which are found on tumor cells,but not to normal cells of the same tissue type. Usually the antibodiesbind to antigens found on a number of tumor cells of different tissuetypes and do not exhibit any substantial binding to a wide variety ofnormal cells. The hybridomas are stable, retaining parental DNA andmaintaining an Ig secretion rate for over a year in culture.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Materials and Methods

Fusion and Selection of Hybridomas

Lymph nodes were teased with nugent forceps in RPMI 1640 media andisolated lymphocytes were cultured overnight at 37° C. and 5% CO₂ inRPMI 1640 with 10% fetal calf serum (FCS) and 2 mM L-glutamine.Lymphocytes were counted and mixed at a ratio of 2:1 with the humanlymphoblastoid B cell line UC729-6 (Handley and Royston, 1982, inHybridomas in Cancer Diagnosis and Treatment, eds. Mitchel and Oettgen,pp. 125-132, Raven Press, NY), then fused with polyethylene glycol 1500by a modification of the technique by Gefter et al., Somatic CellGenetics (1977) 3:321-336. Fused cells were plated at 10⁵ cells/well ina Costar 96 well microtiter plate withHypoxanthine-Amethopterin-Thymidine (HAT, Littlefield, Science (1964)145:709-710) supplemented RPMI 1640 with 10% FCS and L-glutamine. Within10-20 days, wells positive for hybridoma growth were assayed for humanantibody production and their reactivity to a limited human cell panelby an enzyme immunoassay (EIA). Wells positive for reactivity werecloned by limiting dilution without the use of feeder layers andexpanded for further study.

Enzyme Immunoassay

Human MoAbs and their reactivity to cells were detected by amodification by an EIA previously described (Handley et al., J. ofImmunologic Methods (1982) 54:291-296, as modified by Glassy et al., J.Immunologic Methods (1983) 58:119-126). Briefly, 50 μl of either anaffinity purified goat anti-human Ig or a 4×10⁶ target cell/mlsuspension was immobilized in triplicate wells of an immunofiltrationmanifold. (The specially designed microtiter plate which serves as bothan incubation chamber and filtration manifold (VP no. 107) is availablecommercially from V and P Scientific, San Diego, Calif.). The bottom ofeach well contains a 0.6 mm hole over which is placed a 6 mm diameterglass fiber filter. Surface tension prevents fluid volumes less than 100μl from draining through the hole until a vacuum is applied. When vacuumis applied, fluid is drawn through the filter and out the drain holeleaving particulate matter trapped on the filter. After washing 3× with0.3% gelatin in phosphate buffered saline, 50 μl of hybridomasupernatant were incubated 30 min. at room temperature. Filters werethen washed and incubated with 50 μl of a horseradishperoxidase-conjugated goat anti-human Ig for an additional 30 min.Filters were washed again and incubated with 150 μl of a 400 μg/mlsolution of orthophenylene diamine in citrate buffer. 100 μl of eachwell were then transferred to a new plate containing 50 μl of 2.5 M H₂SO₄ and read on a Dynatek (Alexandria, Va.) MR 580 micro-ELISA reader at492 nm.

Hybridoma culture fluids were precipitated with 50% ammonium sulfate andcrude Ig fractions collected. The precipitates were dissolved inphysiological saline and purified by affinity chromatography usingS-aureus Protein A-bound Sepharose with IgG and Sepharose-(sheepanti(humanIgM) antibody) with IgM. From 1L of the culture fluid ofCLNH5, 2.2 mg IgM was obtained, while from 1L of the culture fluid ofCLNH11, 3.0 mg IgG was obtained.

RESULTS

Table 1 outlines the results of the fusion attempting to produceanti-SCCC (squamous cell carcinoma of cervix) human MoAbs. The fusionproducing CLNH5 and CLNH11, human-human hybridomas secreting a MoIgMkand a MoIgG reactive with SCCC cell lines, generated 6 growth positivewells of 80 wells plated. Hybridomas CLNH5 and CLNH11 were cloned andexpanded when found to react with the cervical carcinoma cell lines,CaSki and Hela.

                  TABLE 1                                                         ______________________________________                                        GENERATION AND IDENTIFICATION OF HUMAN MoAbs                                             #Lympho- #Hybri-    #Secret-                                       Lymph Node cytes    domas      ing   #Human                                   draining   fused    generated  M G A reactive                                 ______________________________________                                        Cervical   7.0 × 10.sup.6                                                                   6          2 1 0 2 (CLNH5                                 Carcinoma                            and 11)                                  (SCCC)                                                                        ______________________________________                                    

The relative amounts of human MoAb bound to each of the cell lineslisted was measured by EIA.

Antibody (IgM) secreted by CLNH5 shows positive reactivity withcarcinomas of the cervix (CaSki, Hela), lung (T293, Calu-1, andSk-MES-1), melanoma (SK-MEL-28), and prostate (LnCap) and was negativefor normal fibroblasts, T lymphocytes and peripheral blood lymphocytes.Antibody (IgG) secreted by CLNH11 shows positive reactivity withcarcinomas of the cervix (CaSki, Hela), prostate (PC-3), breast(ZR-76-1), colon (COLO-205) and melanoma (G-361) and was negative fornormal fibroblasts (WI-38 and MRC-9), T. lymphocytes and peripheralblood lymphocytes.

The cytobiochemical properties of the hybridomas of the presentinvention are shown below.

Hybridoma CLNH5:

Hybridoma CLNH5 was deposited at the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209. on Jan.28, 1983 and received ATCC Accession Number HB 8206. Hybridoma CLNH11was deposited at the ATCC on May 20, 1983 and received ATCC AccessionNumber HB 8307.

(1) Number of chromosomes: 60 to 90 (maximum frequency 80).

(2) It secretes human immunoglobulin M (IgM).

(3) Doubling time: 30-40 hours.

(4) Lymphocytic single cell.

(5) Its DNA content is at least two times, for example, 2 to 2.5 times,that of normal human lymphocytes.

(6) IgM binds to human cervical carcinoma cells and the other carcinomasmentioned above.

In addition, the above hybridoma CLNH5 can be proliferated in HAT medium(medium containing hypoxanthine, amethopterin and thymidine).

Hybridoma CLNH11:

(1) Number of chromosomes: 60 to 90 (maximum frequency 80).

(2) It secretes human immunoglobulins G (IgG).

(3) Doubling time: 30-40 hours.

(4) Lymphocytic single cell.

(5) Its DNA content is at least two times, for example 2 to 2.5 times,that of normal human lymphocytes.

(6) IgG binds to human cervical carcinoma cells, and the othercarcinomas mentioned above.

In addition, the above hybridoma CLNH11 can be proliferated in HATmedium.

The relative DNA content (the ratio to the DNA content of normal humanlymphocytes) was determined by a method which comprises dyeing thehybridoma and then analyzing it by a cytofluorometer.

The properties of the monoclonal human immunoglobulins in accordancewith this invention are shown below.

Monoclonal Human Immunoglobulin Produced by the Hybridoma CLNH5

(a) It is human immunoglobulin M (IgM).

(b) It has a stronger binding affinity to cell lines, Hela and CaSki,than to normal fibroblasts (WI-38).

(c) It does not react with human red blood cells, nor shows anagglutination reaction on human red blood cells.

(d) It is composed of heavy chains (H chains) and light chains (Lchains), has a molecular weight of about 180,000 (monomer), and existsas a pentamer in the culture fluid.

Monoclonal Human Immunoglobulin Produced by the Hybridoma CLNH11

(a) It is human immunoglobulin G (IgG).

(b) It has a stronger affinity to cell lines, Hela and CaSki, than tonormal fibroblasts (WI-38).

(c) It does not react with human red blood cells, nor shows anagglutination reaction on human red blood cells.

(d) It is composed of H chains and L chains and has a molecular weightof about 150,000.

The binding activity of human monoclonal antibodies distinguishingneoplastic cells from normal cells was measured as follows:

An original human tissue section including carcinoma cells and normalcells was fixed on a glass plate by glutaraldehyde, and then stained byenzyme immunoassay according to the method of Sternberger et al., J.Hist. Cyto. (1970) 18:315.

Example 2

The fusion and cloning procedure described in Example 1 were used inpreparing the hybridoma described in Example 2. An involved lymph nodefrom a patient who had invasive squamous cell carcinoma of the vulva wasreceived within three hours of surgery. The nodal segments were immersedin serum-free RPMI-1640, trimmed free of extraneous tissue and capsularcomponents, and teased with nugent forcepts to make a single cellsuspension. After large tissue aggregates settled, the cells insuspension were removed and washed twice, pelleting the cells at 500×gfor 5 min/wash. All dissections and cell preparations were performed atroom temperature. The isolated lymphocytes were resuspended at 5×10⁶ /mlin RPMI-1640 media supplemented with 10% fetal calf serum (FCS) andincubated overnight at 37° C. in 5% CO₂ prior to fusion.

Using 35% polyethylene glycol 1500, 3.33×10⁷ of the patient'slymphocytes were fused with 1.66×10⁷ UC 729-6 cells according to theprocedure described above. After the fusion, the cells were added to96-well plates at 1.0×10⁵ cells per well. The day after the fusion, thegrowth media was replaced by RPMI-1640 media supplemented with 10% FCSand glutamine, including hypoxanthine (1×10⁻⁴ M), amethopterin (2×10⁻⁷M, and thymidine (1.6×10⁻⁵ M) (HAT media)(13). After 2-4 weeks inculture, hybrids visible to the eye were analyzed for human Igsecretion. Eighty hybrids resulted from 480 wells plated, 14 of whichsecreted IgG and four of which secreted IgM.

The hybridoma supernatants were screened as in Example 1. Those whichsecreted Ig were expanded and cloned by limiting dilution without anyfeeder layer cells in RPMI media.

Cryopreservation and thawing of these hybridomas did not result in anydetectable adversity in stability and secretion. The hybrids generatedwere tetraploid as judged by karyotypic and cytofluorographic analysisof their DNA content (Table 2).

                  TABLE 2                                                         ______________________________________                                        CYTOFLUOROGRAPHIC ANALYSIS OF HUMAN                                           HYBRIDOMA DNA                                                                        Relative DNA Content.sup.a                                             Cell Type                                                                            2 months  4 months 8 months                                                                             12 months                                                                            24 months                             ______________________________________                                        UC 729-6                                                                             45 ± 2 44 ± 2                                                                              45 ± 4                                                                            45 ± 2                                                                            45 ± 2                             VLN3G2 80 ± 2 81 ± 3                                                                              80 ± 4                                                                            80 ± 3                                                                            N.D.                                  VLN1H12                                                                              82 ± 3 81 ± 3                                                                              81 ± 3                                                                            80 ± 3                                                                            N.D.                                  CLNH5.sup.b                                                                          83 ± 3 82 ± 2                                                                              83 ± 3                                                                            83 ± 3                                                                            82 ± 3                             MHG7.sup.c                                                                           74 ± 2 68 ± 3                                                                              55 ± 3                                                                            54 ± 3                                                                            51 ± 2                             ______________________________________                                         .sup.a Relative DNA content of UC 7296 and the hybridomas was obtained by     the propidium iodine method (Glassy et al., Proc. Natl. Acad. Sci. USA        (1983) 80:6327). At the indicated times after fusions, the DNA content of     the cells was determined.                                                     .sup.b CLNH5 is described in Example 1.                                       .sup.c MHG7 is a mousehuman hybridoma described in Lowe et al., J. Urol.      (1984) 132:780.                                                          

HLA phenotyping has also shown that these human hybridomas expressantigens of both parental UC 729-6 and the patient's lymph nodelymphocytes (Table 3).

                  TABLE 3                                                         ______________________________________                                        HLA PHENOTYPE OF UC 729-6 AND VLN HYBRIDS                                     Cell Line   HLA-A     HLA-B       HLA-DR                                      ______________________________________                                        UC 729-6    A1, A2    B5, B17     DR4, DR7                                    VLN3G2      A1, A2    B5, B17     DR4, DR7                                                          B27, BW44                                               VLN1H12     A1, A5    B5, B17     DR4, DR7                                                          B27                                                     ______________________________________                                    

Because large amounts of antibody would be required in a clinicalsetting, the long-term stability of human Ig-secreting hybridomas wasstudied. The issue of long-term stability of these VLN hybrids wasassessed over a 12-month period, during which both DNA content (Table 2)and Ig secretion rate (Table 4) were examined. The generated dataindicated that UC 729-6 is a genetically stable vector for the captureand immortalization of human B lymphocytes.

                  TABLE 4                                                         ______________________________________                                        IgM SECRETION (μg IgM/10.sup.6 cells/ml/day) BY                            HUMAN HYBRIDOMAS OVER TIME                                                    Time                                                                          (Months)    VLN1H12     CLNH5     MHG7                                        ______________________________________                                        1           1.58        2.36      2.84                                        2           ND          2.48      2.60                                        3           1.37        ND        1.92                                        4           ND          2.28      1.45.sup.a                                  5           ND          ND        2.11                                        6           1.25        2.43      1.87                                        7           ND          ND        1.49                                        8           1.29        2.36      ND                                          9           ND          ND        1.16.sup.a                                  10          1.31        2.43      2.48                                        11          ND          ND        2.27                                        12          1.28        2.24      2.06                                        ______________________________________                                         At the indicated time points after the fusion, the amount of human IgM in     the culture supernatant was determined by EIA. ND = not determined.           .sup.a MHG7, a mousehuman hybridoma described in Table 2, was subcloned b     limiting dilution (1 cell/3 wells) at 4 and 9 months postfusion to select     for a higher secreting subclone.                                         

Immunoreactivity

Of the 14 IgG-secreting hybrids, six passed initial reactivity screensagainst target cell bound antigens, whereas one of the fourIgM-secreting hybrids passed. These seven hybridomas were cloned bylimiting dilution (1 cell/3 wells) and expanded for further analysis.The reactivity index (R1) of the six IgG human MoAbs against a panel ofnormal and malignant cells was calculated from the following formula:##EQU1## The control consisted of an irrelevant human IgG used at thesame concentration as the test human MoAb.

These six IgG MoAbs have broad reactivity profiles with human carcinomacells, but do not react with hematopoietic or fibroblast cell lines andnormal peripheral blood leukocytes. Of particular note, all six of theIgGs were very reactive with the A431 cell line, a carcinoma of thevulva, similar to the primary cancer of the patient used in thesestudies.

A titration of the VLN3G2 and VLN5C7 IgG MoAbs against target cell-boundantigens was determined. Starting from an initial concentration of 450ng/ml of VLN3G2 appreciable reactivity was obtained even at 450 pg/ml.There did not appear to be any major quantitative differences in theantigen expression by either the vulva, lung, or stomach cell linessince the reactivity of the MoAbs was similar in each case.

Target A431 cells were analyzed by indirect immunofluorescence withVLN3G2. Cell surface and cytoplasmic staining patterns were evident.

Example 3

The hybridomas described in Example 3 were prepared in the same manneras the hybridomas of Example 1. The hybridoma designated WLNA6 wasprepared from lymphocytes isolated from a lymph node draining a Wilm'stumor. A Wilm's tumor is a malignant renal tumor which has frequentlymetastasized to the lungs or elsewhere when a renal mass is located.

CLNH5 and WLNA6 were studied because the supernatants reacted with anumber of cell lines (Table 5).

                                      TABLE 5                                     __________________________________________________________________________    REACTIVITY OF HUMAN MoAbS WITH HUMAN CELL LINES                                             OD.sub.490                                                      Cell Line                                                                          Type     8A1     CLNH5  WLNA6                                            __________________________________________________________________________    Calu-1                                                                              Lung carcinoma                                                                        0.031 ± 0.008                                                                      0.342 ± 0.061                                                                     0.029 ± 0.009                                 Sk-MES-1                                                                           Lung carcinoma                                                                         0.025 ± 0.007                                                                      0.395 ± 0.054                                                                     ND                                               T293H                                                                              Lung carcinoma                                                                         0.011 ± 0.005                                                                      0.498 ± 0.081                                                                     0.753 ± 0.225                                 HeLa Cervical carcinoma                                                                     0.029 ± 0.010                                                                      0.443 ± 0.075                                                                     0.140 ± 0.041                                 CaSki                                                                              Cervical carcinoma                                                                     0.021 ± 0.009                                                                      0.429 ± 0.084                                                                     0.145 ± 0.073                                 Ln-Cap                                                                             Prostate carcinoma                                                                     0.033 ± 0.007                                                                      0.386 ± 0.041                                                                     0.005 ± 0.003                                 350Q Normal fibroblast                                                                      0.040 ± 0.011                                                                      0.026 ± 0.008                                                                     0.041 ± 0.014                                 WI-38                                                                              Normal fibroblast                                                                      0.034 ± 0.088                                                                      0.038 ± 0.009                                                                     0.033 ± 0.015                                 __________________________________________________________________________     OD.sub.490 values were read on a Dynatech microELISA reader (model MR 580     in a quantitative immunofiltration assay (23). Values represent mean ±     SD of triplicate determinations. ND, not determined.                     

The CLNH5 IgM MoAb reacted with several human malignant cell lines ofcervical, lung, and prostate origin but not with normal humanfibroblasts. The WLNA6 IgM MoAb reacted strongly with T293, an oat cellcarcinoma of the lung, had weak reactivity with the HeLa and CaSkicervical carcinoma cell lines, and had no reactivity with lung carcinomaCalu-1, prostate carcinoma Ln-Cap, or WI-38 and 350Q normal fibroblasts.The 8A1 IgM MoAb failed to react with any of the tested cell lines andserved as a negative control. 8A1 is a hybridoma produced from thefusion UC 729-6 and the peripheral blood lymphocytes of a patient withchronic lymphocytic leukemia.

Example 4

As described previously, human lymphocytes obtained from regionaldraining lymph nodes of patients with cancers of the cervix, kidney andvulva were fused with a human fusion partner, UC729-6, to producehybridomas designated CLNH5, WLNA67 and VLN1H:12, respectively. Humanlymphocytes from a regional hymph node of a patient with prostate cancerwas fused to a mouse fusion partner, P3-NS-1-Ag4-1, using the samefusion protocol.

The hybridomas and their secreted antibodies were analyzed to determinethe genetic stability of the hybridomas. The following materials andmethods, in addition to those previously described, were used.

Cell Lines

The following human cell lines were used in Example 4. Leukemia celllines, Molt-4, CEM, HPB-A11, K562, ML-3, KG-1, and 8402; melanoma celllines, M21, MEW0, SK-MEL-28; macrophage cell line U937 (kindly providedby Dr. K. Nilsson); normal fibroblasts, 350Q, WI-38; colon carcinomacell lines, HT-29, T-84; kidney cell line, Caki-2; carcinomas of thebladder, T-24, Scaber; stomach carcinoma cell lines, AGS-10, AGS,Kato-III, MKN-74, MKN-28; vulva carcinoma cell line, A431; ovariancarcinoma cell line, SK-OV-3; prostatic carcinoma cell lines, DU 145,PC-3, Ln-Cap; cervical carcinoma cell lines, Hela and Caski; murinemyeloma P3-NS-1-Ag4-1; and murine IT22 fibroblasts.

Cell Surface Phenotyping

Cell surface phenotypes of UC 792-6, MHG7, CLNH5, and VLN1H12 wereanalyzed by indirect immunofluorescence using procedures and reagentspreviously described in Royston et al., J. Immunol. (1980) 125:715.

Cytofluorographic Analysis of DNA Content

Cells and hybridomas were stained by the propidium iodide method for thedetermination of relative cell DNA content and analyzed bycytofluorometry, as described in Taylor, J. Histochem. Cytochem. (1980)28:1021. Briefly, 1.0×10⁶ cells were fixed in 70% ethanol for 2 hours,washed (500×g for 10 minutes), and suspended in 100 μl PBS. Onemilliliter of propidium iodide (0.05 mg/ml) (Sigma) was added to thecell suspension, incubated for 15 minutes at 4° C., and filtered througha 50 μm pore nylon mesh. Samples were analyzed on an OrthoCytofluorograf 50H with computer 2150 equipped with a 488 mm argon laserat 400 mW. All data were generated by the computer.

Isozyme and Karyotype Analysis

The presence of unique isozyme patterns from human chromosomes weredetermined by established procedures (Glassy et al., Cancer Res. (1982)42:3971; Kamarck et al., Exp. Cell Res. (1984) 152:1). Human chromosome2 was identified by malate dehydrogenase (E.C. 1.1.1.37) and humanchromosome 14 was identified by nucleoside phosphorylase (E.C. 2.4.2.1).

The antibody secretion rates of the human-human hybridomas was comparedto that of the mouse-human hybridoma over a 12-month period. IgMsecretion of CLNH5 and VLN1H12 was relatively stable at 2.5 μg/ml and1.5 μg/ml, respectively, over the time period indicated. MHG7, the mousehuman hybrid, however, required subcloning at three and eight months inculture to retain antibody secretion. Cultures of MHG7 not subclonedroutinely lost antibody production entirely. Subcloning of MHG7generated clones of greater antibody production capability and clonesentirely lacking antibody production. Isoenzyme analysis of somesubclones of MHG7 (Table 6) revealed the presence of both humanchromosomes 2 and 14 in those producing human IgM whereas those cloneslacking either chromosome 14 or 2 and 14 produced no detectable IgM.Human chromosomes 2 and 14 are known to contain the loci for kappa lightchain (McBride et al., J. Exp. Med. (1982) 155:1480) and Ig heavy chain(Croce et al., Acad. Sci (USA) (1979) 76:3416), respectively.

                  TABLE 6                                                         ______________________________________                                        Isoenzyme Analysis of MHG7                                                    Human                          Amount Human                                   Chromosome         MHG7        IgM Secreted                                   2      14          Subclone    (ng/ml)                                        ______________________________________                                        -      -           MHG7M.1.1    <32.sup.a                                     +      -           MHG7M.1.2    <32                                           +      +           MHG7M.1.3   1850                                           +      +           MHG7M.1.4   1100                                           -      +           MHG7M.1.5    <32                                           ______________________________________                                         .sup.a The lower limit of the EIA used was 32 ng/ml. Midlog phase cells       were harvested from culture and somatic cell hybrid isoenzymes were           characterized electrophoretically on cellulose acetate gels according to      standard procedures (Glassy et al., Cancer Res. (1982) 42:3971; Kamarck e     al., Exp. Cell. Res. (1984) 152:1) and analyzed by EIA for the amount of      human IgM secreted (see Materials and Methods).                          

Relative DNA contents or the human Ig secreting hybridomas are shown inTable 7.

                  TABLE 7                                                         ______________________________________                                        Cytofluorographic Analysis of Human Hybridoma DNA                                     Relative DNA Content.sup.3                                            Cell Type                                                                             2 mo     4 mo     8 mo   12 mo  24 mo                                 ______________________________________                                        UC 729-6                                                                              45 ± 2                                                                              44 ± 2                                                                              45 ± 2                                                                            45 ± 2                                                                            45 ± 2                             CLNH5   83 ± 3                                                                              82 ± 2                                                                              83 ± 3                                                                            83 ± 3                                                                            83 ± 3                             VLN3G2  80 ± 3                                                                              81 ± 3                                                                              80 ± 4                                                                            ND     ND                                    MHG7    74 ± 2                                                                              68 ± 3                                                                              55 ± 3                                                                            54 ± 3                                                                            51 ± 2                             VLN1H12 82 ± 3                                                                              81 ± 3                                                                              81 ± 3                                                                            ND     ND                                    ______________________________________                                         .sup.a Relative DNA contents of UC 7296, humanhuman hybrid MHG7 were          obtained by the propidium iodide method (Taylor. J. Histochem. Cytochem.      (1980) 28:1021). At the indicated times after fusions, the DNA contents o     the cells were recorded.                                                      ND = not determined.                                                     

Over a two-year period, the tetraploid hybridoma CLNH5 and theheteroploid mouse-human hybrid MHG7 were compared with the diploid UC729-6. The DNA contents of CLNH5 and UC 729-6 have been relativelystable over 24 months in culture while MHG7 had lost a significantnumber of chromosomes over the same period of time.

The surface marker phenotype of UC 729-6 and hybrids are shown in Table8.

                                      TABLE 8                                     __________________________________________________________________________    Cell Surface Phenotypes.sup.a                                                 Phenotypic                                                                          % Positive Cells                                                        Marker                                                                              Function UC 729-6                                                                            MHG7 CLNH5                                                                              VLN1H12                                        __________________________________________________________________________    Control                                                                             --       3     12   2    2                                              T101  pan T-cell                                                                             2     11   2    2                                              Leu4  pan T-cell                                                                             4     13   2    3                                              Leu3  helper T-cell                                                                          4     11   2    3                                              Leu2  suppressor T-cell                                                                      4     15   3    5                                              I.sub.2                                                                             HLA-DR   97    12   98   100                                            BA1   pan B-cell                                                                             7     11   3    4                                              BA2   hematopoietic                                                                          4     10   62   67                                                   precursor                                                               BA3   common ALL                                                                             4     11   1    3                                              sIg   immunoglobulin                                                                         +     +    +    +                                              __________________________________________________________________________     .sup.a Mid logphase cells were harvested from culture and analyzed on an      Ortho Cytofluorograf 50 H and computer system 2150 using reagents and         procedures previously described in Royston et al., J. Immunol. (1980)         125:715. sIg was assessed using indirect immunofluorescence under a           fluorescence microscope (Royston et al., supra).                         

The most significant finding was the expression on the human hybrids ofthe BA2 antigen associated with precursor hematopoietic cells. Thisantigen was not expressed on UC 729-6.

Reactivity of Human Monoclonal IgM

The reactivity of WLNA6 with a panel of human cell lines was determined.Five other human IgM monoclonals generated in the same fusion as WLNA6,and deemed of interest in an initial screen, failed an expanded screen.WLNA6 was reactive with 3 of 15 cell lines tested, the highest activitybeing T293H, a squamous cell carcinoma of the lung.

The reactivity of the monoclonal IgM antibodies from CLNH5, VLN1H12, andMHG7 with a larger panel of cell lines was determined. The reactivityindex was calculated as follows:

    RI=O.D.490 test MoAb-O.D.490 background/O.D.490 irrelevant MoIg-O.D.490 background

By these criteria, CLNH5 was reactive with 8 of 31 cells tested; thesebeing carcinomas of the cervix (CaSki, Hela), lung T293H, Calu-1, andSK-MES-1), melanoma (SK-MEL-28), prostate (LnCap), and vulva (A431),VLN1H12 was reactive with 15 of 27 cell lines tested; carcinomas of thecervix (CaSki, Hela), kidney (Caki-2), lung (NCI-69, T293H), melanoma(SK-MEL-28), pancreas (Capan-1), prostate (LnCap, PC-3, DU-45), stomach(MNK-28, KATO-III, AGS-10, AGS), and vulva (A431) were all reactive.MHG7 was reactive with 8 of 37 cell lines tested; carcinomas of thecolon (T-84), lung (T293H, T222, T291), prostate (LnCap, PC-3), stomach(Kato-III), and vulva (A431) were positive.

Titrations of MHG7, CLNH5, and VLN1H12 against cell-bound targetantigens are shown in Table 9. With as few as 2×10³ target cells/well,the EIA detected as little as 20 ng of cell-bound human IgM MoAb.

                  TABLE 9                                                         ______________________________________                                        Titration of Human Monoclonal Antibodies                                      Against Cell-Bound Target Antigens.sup.a                                      MoAb Cell Line.sup.b                                                          MHG7   Ln-Cap   CLNH5     CaSki  VLN1H12 A431                                 ______________________________________                                        3100.sup.c                                                                           0.465    2000      0.441  1450    0.325                                 620   0.396    400       0.373  290     0.174                                 124   0.271    80        0.229  58      0.171                                 25    0.121    16        0.118  12      0.146                                  5    0.095    3.2       0.084  3       0.144                                  1    0.102    0.64      0.084  1.5     0.149                                Background: 0090                                                                          Background: 0.085                                                                            Background: 0.145                                  ______________________________________                                         .sup.a Cells were used in the EIA at 2.0 × 10.sup.3 cells/well. (Se     Materials and Methods).                                                       .sup.b The cell line used with each MoAb is allogeneic with the patient's     cancer from which the lymph nodes were obtained. MHG7 was derived from        prostate cancer and LnCap is a prostate carcinoma cell line. CLNH5 from       cervical cancer and CaSki is a carcinoma of the cervix; VLN1H12 from vulv     cancer and A431 is a carcinoma of the vulva.                                  .sup.c Concentration of human IgM is expressed in ng/ml.                 

The subject monoclonal antibodies are useful for diagnosing, imaging andpotentially for treating cervical carcinoma as well as other reactivetumors. Because of the specificity of the monoclonal antibodies over arange of cervical carcinomas from different hosts, the subjectantibodies can be used in different hosts, rather than solely with thehost source of the antigen. Similarly, the monoclonal antibodiesfrequently are useful to identify carcinomas of a number of tissue typesin addition to the type of tumor of the host source of the lymphocytes.Because the subject antibodies are human, they are less likely toproduce a significant immune response when employed in in vivo diagnosisor therapy.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. An antibody composition comprising aphysiologically acceptable carrier and a monoclonal antibody having thesame binding specificity as that of the monoclonal antibody expressed bycell line CLNH11, ATCC Accession No: HB8307.
 2. The composition of claim1, wherein said antibody is labeled with a label capable of providing adetectable signal.
 3. The composition of claim 1, wherein said antibodyis labeled with a toxin.
 4. The composition of claim 1, wherein saidantibody is labeled with a radionuclide.
 5. The composition of claim 1,wherein said antibody is attached to a liposome.
 6. The composition ofclaim 1, wherein said antibody is an antibody expressed by cell lineCLNH11, ATCC Accession No: HB8307.
 7. An antibody composition comprisinga physiologically acceptable carrier and a monoclonal antibody fragmenthaving the same binding specificity as that of the monoclonal antibodyexpressed by cell line CLNH5, ATCC Accession No: HB8206.
 8. Thecomposition of claim 7, wherein said antibody is labeled with a labelcapable of providing a detectable signal.
 9. The composition of claim 7,wherein said antibody is labeled with a toxin.
 10. The composition ofclaim 7, wherein said antibody is attached to a liposome.
 11. Thecomposition of claim 7, wherein said antibody is attached to aradionuclide.
 12. The composition of claim 7, wherein said antibody isan antibody expressed by cell line CLNH5, ATCC Accession No: HB8206.